The objectives of the research are to investigate physical processes by which polytopic integral membrane protein sequences are inserted spontaneously into and oriented properly within membranes. It is hypothesized that insertion and topogenesis processes are controlled not only by distributions of positively charged amino acids across the transmembrane segments of polytopic proteins, but also by 1) topogenic sequence interactions within insertion elements referred to as helical hairpins, and 2) structural constraints imposed on transmembrane segment orientations by multiple segment and loop interactions that occur within these proteins. The research will investigate the requirement for specific sequence interactions between transmembrane segments of helical hairpins during insertion, and the requirement for defined polar loop sequences for hairpin insertion into membranes. The dynamics of transmembrane segment movements occurring within hairpins during insertion also will be investigated. Lastly, the locations of putative positive charge-independent auxiliary topology control sequences referred to as topological anchoring sequences and topogenic interaction sequences will be identified. These sequences will be assayed for their ability to function in the absence of positive charges. Research will be conducted with membrane insertion mutants of the Escherichia coli integral membrane transport protein known as the tetracycline resistance protein (TetA). TetA proteins catalyze expulsion of tetracycline antibiotics from the bacterial cytoplasm, and are responsible for resistance of many pathogenic bacteria to these drugs. Investigation of TetA topogenesis will be performed by proteolysis of fusion proteins between E. coli maltose-binding protein (MBP) and TetA, in which MBP serves as an immunological tag for detecting the protein. Knowledge learned will be important toward solving the mechanism by which polytopic integral membrane proteins insert into the membrane. It also will uncover important structural interactions occurring within TetA, and by extension, within proteins belonging to a large family of related transporters known as the major facilitator superfamily. Ultimately, information about the structure of TetA may be useful in designing inhibitors of its transport activity.